Bladder cancer is a common malignancy with approximately 55,000 new cases and nearly 12,000 deaths reported in the US annually. Although a high proportion of patients (65-70 percent) present with well or moderately well differentiated superficial transitional cell carcinomas, approximately 60-70 percent of these nevertheless go on to develop recurrent disease after resection of primary lesions. For this reason, surgery is often combined with various adjuvant therapies including intravesical chemotherapy and/or immunotherapy (i.e. BCG). While such treatments do improve control, relapses remain common encouraging consideration of various alternative approaches including gene therapy. The major objective of this proposal is to evaluate a novel gene therapy-based approach to the treatment of bladder cancer in which alternative splicing is used as a means of targeting the expression of the enzyme alkaline phosphatase (ALP) to tumor cells in vivo. Specifically, adenoviral vectors will be constructed in which the expression of ALP is dependent upon the accurate removal from chimeric pre-mRNA transcripts of alternatively spliced intronic sequences. These vectors will be used to confirm the targeting specificity of alternative splicing in vitro following transduction of a panel of bladder tumor cell lines that differ in expression of various alternatively spliced isoforms of the adhesion protein CD44. Adenoviral vectors will also be tested both in vitro and in an ex vivo explant model for their ability to sensitize bladder tumor cells that differ in alternative splicing ability, to killing by the inactive prodrug Etopophos, which is converted to the potent topoisomerase II inhibitor etoposide upon dephosphorylation by ALP. Finally, in order to better identify those patients most likely to benefit for this targeted therapy, studies will be initiated to define the transacting factors and cis-acting sequences that regulate the alternative splicing of CD44 in bladder tumor cells.